Regulation of D-amino acid oxidase expression in the yeast Rhodotorula gracilis

Yeast. 2003 Sep;20(12):1061-9. doi: 10.1002/yea.1023.

Abstract

Rhodotorula gracilis is a oleaginous yeast which utilizes D-amino acids as a source of carbon and/or nitrogen. D-amino acid oxidase (DAAO), which converts D-amino acids in the corresponding alpha-keto acids and ammonia, is the first enzyme involved in the catabolism of D-amino acids. DAAO activity is induced by the presence of D-alanine, but the presence of the L-isomer prevents induction by inhibiting the transport of D-alanine into cells. To understand how DAAO expression is regulated, R. gracilis cells were grown on media containing different nitrogen and/or carbon sources. As a general rule, the level of DAAO mRNA reached a maximum after 15 h growth and preceded by approximately 6 h the maximum level of DAAO activity. The inducer D-alanine acts by increasing the rate of DAAO mRNA transcription: the increase in DAAO expression is due essentially to de novo synthesis. The presence of a supplemental carbon source (e.g. succinate or glucose) does not repress DAAO expression. Ammonium sulphate appears to have a negative effect on DAAO mRNA translation and on the expression of DAAO activity: DAAO is only partially active when the yeast is grown in the presence of D-alanine and ammonium sulphate. The best expression of DAAO activity was obtained by growing the cells for 12 h at 30 degrees C in the presence of glucose and D-alanine using cells pre-cultured for 10 h on glucose and L-alanine (0.99 U/mg protein, corresponding to approximately 1.0% total proteins in the crude extract). Under these growth conditions a six-fold increase in DAAO production was achieved.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / metabolism
  • Ammonium Sulfate / metabolism
  • Blotting, Northern
  • Blotting, Western
  • D-Amino-Acid Oxidase / biosynthesis*
  • D-Amino-Acid Oxidase / genetics
  • D-Amino-Acid Oxidase / metabolism
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Expression Regulation, Enzymologic / physiology*
  • Gene Expression Regulation, Fungal / physiology*
  • Glucose / metabolism
  • RNA, Fungal / biosynthesis
  • RNA, Fungal / genetics
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Rhodotorula / enzymology
  • Rhodotorula / genetics
  • Rhodotorula / physiology*

Substances

  • Fungal Proteins
  • RNA, Fungal
  • RNA, Messenger
  • D-Amino-Acid Oxidase
  • Glucose
  • Alanine
  • Ammonium Sulfate